key: cord-0827392-8zn6pnu2 authors: Fesseha, Haben; Mathewos, Mesfin; Aliye, Saliman; Mekonnen, Endale title: Isolation and antibiogram of Escherichia coli O157: H7 from diarrhoeic calves in urban and peri‐urban dairy farms of Hawassa town date: 2021-12-15 journal: Vet Med Sci DOI: 10.1002/vms3.686 sha: 8d5bfc1f69b672309657f09945fa25188f55c9c3 doc_id: 827392 cord_uid: 8zn6pnu2 BACKGROUND: Calf diarrhoea is the most serious issue in the livestock industry, resulting in significant financial losses. METHODS: A study was undertaken in 32 urban and peri‐urban dairy farms of Hawassa town to isolate E. coli from diarrhoeic calves, assess associated putative factors related to the occurrence, and the evaluate antibacterial susceptibility patterns of isolates. A convenience sampling technique was performed for the selection of these dairy farms and calf samples. A total of 68 faecal samples were collected directly from the rectum of diarrhoeic calves. The faecal samples were confirmed as E. coli O157: H7 positive using the latex agglutination test. RESULTS: In this study, 47(69.1%) samples were positive for E. coli, of which 22 (46.8%) were identified as E. coli O157:H7 strains based on their latex agglutination character. Factors such as frequency of calf house cleaning, type of supplement provided, and method of colostrum feeding were significantly correlated (p < 0.05) with calf diarrhoea, while the other risk factors had no significant association. Antibiogram of E. coli O157:H7 isolates showed that the isolates were highly sensitive to gentamycin, ceftriaxone, trimethoprim‐sulphamethoxazole, and ciprofloxacin and were found to be resistant to tetracycline, kanamycin and amoxicillin. CONCLUSION: Our findings revealed that calf diarrhoea is still a major health problem of calves in the study area. Hence, improved calf and farm management practice, an ad libitum quantity of colostrum, and good farm hygienic practices should be ensured. This study also revealed that some antibiotic‐resistant E. coli O157:H7 isolates need to be further investigated for their public health implications. Ethiopia is the largest livestock resourceful country on the African continent, with approximately 57.8 million cattle, 29.33 million sheep and 29.11 million goats (Central Statistical Agency, 2015) . In addition, this sector contributes 16.5% of the national Gross Domestic Product (GDP) and 35.6% of the agricultural GDP (Behnke, 2010; Metaferia et al., 2011) . Dairying is a form of livestock production that is practiced almost everywhere in the world, including Ethiopia, and involves a large number of small-, medium-or large-scale, subsistence or marketoriented farms, with herd size being the primary determinant. It is the primary source of income for dairy farmers in Ethiopia's urban and periurban settlements (Chagunda et al., 2006; Hadgu et al., 2021) . Newly born calves are a significant source of livestock production for beef and breeding worldwide. However, calves have encountered many complications, such as diarrhoea, pneumonia, joint disorders, umbilical infections, trauma and congenital abnormalities (Heinrichs & Radostits, 2001) . Calf morbidity and mortality were ranked next to mastitis as the second largest problem for dairy production in Ethiopia (ILCA, 1994) . The mean annual birth-to-weaning mortality in calves was reported to be in the range of 2-18.5% in the mixed crop-livestock system in different parts of Ethiopia (Fentie et al., 2016; Fentie et al., 2020; Hadgu et al., 2021) . Non-infectious causes such as trauma to the different parts of the gastrointestinal tract (hardware disease due to metallic foreign bodies, oesophageal and bowel obstruction such as intussusception and volvulus due to various types of foreign bodies), metabolism (simple indigestion, ruminal acidosis and alkalosis) (Fubini & Divers, 2008) , malnutrition, other non-specific or miscellaneous causes, and poor husbandry practices are responsible for calf diarrhoea as well as calf mortality and morbidity in general in different parts of Ethiopia (Hadgu et al., 2021; Romha, 2014) . One of the most common causes of calf mortality and morbidity in the dairy industry is neonatal calf diarrhoea or scour (Fentie et al., 2020; Hadgu et al., 2021; Tajik et al., 2012) . Calf diarrhoea is caused by a variety of infectious (bacteria, viruses and parasites) and noninfectious agents (poor quality and quantity of colostrum, environmental stress due to extreme weather and poor husbandry) (Cho & Yoon, 2014) , especially in calves under the age of 12 days (El-Seedy et al., 2016) . Some of these infectious agents have been linked to food-borne disease zoonosis in humans (El Ayis et al., 2015) . Bovine rotavirus (BoRV), Cryptosporidium parvum, Bovine coronavirus (BoCV), Salmonella, and E. coli are some of the most common enteropathogens that cause calf diarrhoea (Meganck et al., 2015) . Although E. coli is a minor pathogen in most studies of developed countries, such as the United States and Australia, in Ethiopia, it is among the major pathogens in most dairy farms due to poor hygienic conditions and the handling of feeding utensils, calving areas and calf pens (Gebregiorgis & Tessema, 2016; Kidane, 2014; Mohammed et al., 2020; Yeshiwas & Fentahun, 2017) . E. coli is a cause of calf diarrhoea, also known as white scour (Kolenda et al., 2015) , and causes diarrhoea, haemorrhagic colitis, and dysentery in weak, malnourished, debilitated and immunosuppressed calves, particularly calves that do not receive maternal antibodies through colostrum feeding (Ellaithi, 2004; Mailk et al., 2013; Mohamed, 2009 (Bazeley, 2003) . Treatment costs, time spent on medication and resulting chronic illness, thrift and reduced growth efficiency, loss of genetic capacity both from the loss of the calf and the farmer's unwillingness to invest in higher priced semen in the face of a calf mortality crisis and impair adequate heifer replacement are all factors that contribute to economic losses (Bazeley, 2003) . Heifer substitution has a significant impact on dairy farmers' ability to maximise productivity by enabling them to cull low-producing cows selectively. Calf mortality is a major economic problem for all dairy farmers, especially those who farm intensively (Moran, 2011) . E. coli is a Gram-negative, rod-shaped, flagellated, non-sporulating and facultative anaerobic bacterium of the family Enterobacteriaceae. It is the most genetically versatile bacteria and supplies both plasmid and phage-mediated genes. E. coli produces septicaemia and diarrhoea in a wide range of hosts including humans and animals (Hemashenpagam et al., 2009 (Bartels et al., 2010; Kolenda et al., 2015; Moxley & Smith, 2010) . Escherichia coli O157: H7 is the most important bacterial pathogen that causes life-threatening infections such as haemorrhagic colitis (HC), abdominal pain, bloody diarrhoea, haemolytic uremic syndrome and kidney failure, particularly in humans worldwide (Mersha et al., 2010; Pal et al., 2016) . Milk and other dairy products are mostly contaminated with E. coli O157: H7 during direct exposure to faeces due to poor handling systems and cause intestinal or extra-intestinal disease (Bacon et al., 2000; Bélanger et al., 2011) . The high prevalence of E. coli O157: H7 in dairy products may be due to improper milking hygiene, poor house hygiene, lack of post milking teat dipping and practicing of milk by contact labour use of lubricants, and absence of order in milking cows of different ages. Moreover, its occurrence was high in dairy farms without noticeable farm treatment (Radostits et al., 2016) . Antibiotic-resistant strains of this bacterium cause longer and more serious diseases in susceptible animals. Numerous studies have shown that the impact of E. coli on antibiotic resistance increases over time for various antimicrobials (Cortés et al., 2010; Orden et al., 2000; Tadesse et al., 2012) . Inappropriate or irrational use of antimicrobial drugs against diarrhoeal infection in humans and animal has presumptively assumed possible causes of antibiotic resistance. This may pose a F I G U R E 1 Geographical location of Hawassa, ArcGIS software, 2018 possible health danger to humans and animals with regard to the growth of resistant bacterial strains and drug residues. With certain scientific expertise and antibiotic profiles of various diarrhoeal bacterial isolates, effective therapeutic steps for managing diarrhoea of calves have to be regularly studied (Kaura et al., 1988) . In Ethiopia, particularly in Hawassa city, the isolation, identification of E. coli from diarrhoeic calves and evaluation of antibacterial sensitivity patterns of E. coli have not been widely studied. As a result, the study aimed to isolate E. coli, assess antibiotic susceptibility patterns and determine the factors that contribute to the occurrence of E. coli from a diarrhoeic calf in Hawassa City's urban and peri-urban areas. The current study took place at selected dairy cattle farms in Hawassa The study animals were local and Holstein Friesian cross-breed calves of both sexes up to 3 months of age that were kept under different management systems (extensive, semi-intensive and intensive) and clinically affected with diarrhoea and exhibited indicators of systemic disease (e.g., low appetite, fever, dehydration, weakness and impaired suckling reflex) and excreted different types of diarrhoea having diverse colours. The faecal consistency or type of diarrhoea was classified into watery (presence of profuse water-logged faecal particles), bloody (faeces with blood or blood clots), mucoid (presence of viscous mucous within the faecal) and mixed (presence of blood or particles of undigested food, blood clots or pieces of intestinal tissue) (Graham et al., 2018; Renaud et al., 2020) . During the study, 32 dairy farms were selected using convenience sampling from total dairy farms in the study areas on the basis of accessibility and willingness of the farm owners to participate in the study and grouped into smallholder (≤5 heads of milk cow), medium-sized farms (6-50 heads of milk cow) and large-scale (>50 heads of milk cattle) farms depending on the number of cows available in the selected farms (Lema et al., 2001) . In addition, 15 small, 15 medium and 2 largescale dairy farms located in urban and peri-urban areas were involved in the study. The study farms practice semi-intensive and intensive management systems. The ages of diarrhoeic calves were categorised into five age groups: 1-7 days, 8-15 days, 16-30 days, 31-60 days and 61-90 days according to Gebregiorgis and Tessema (2016) . Different factors related to the onset of diarrhoea, such as floor type, the practice of having a separate pen and colostrum feeding time and its duration, were recorded before samples were collected. A cross-sectional study design was employed from October 2019 to May 2020 in large-, medium-and small-scale dairy farms in Hawassa city and its surrounding farms. Non-probability convenience and purposive sampling were used for the selection of farms and faecal samples from each diarrhoeic calf. Thus, a total of 68 diarrhoeic calves (aged between 1 and 90 days) were sampled based on convenience sampling. In addition, factors such as the proportion of the calf population on each farm, case availability and the willingness of the farm owners were considered during the study. The health status of each calf was evaluated through a detailed clinical examination using different types of clinical signs and parameters. Calves that showed poor appetite, rough hair coat, fever, dehydration, sunken eye, reduced suckling reflex, non-treated, weakness and abnormal faecal consistency (diarrhoea) were considered for the present study. The dairy owners were given a semi-structured questionnaire to evaluate the overall husbandry of calves via face-to-face discussions. Generally, the questionnaire contains the following sorts of diarrhoea: calf health, hygiene, health issues, colostrum feeding times and periods. In addition to the surveys, housing, farm hygiene and barn floor direct observational evaluations were carried out. Housing hygiene was graded from 1 to 4: 1 -very clean, 2 -clean, 3 -poor and 4 -very poor according to the previous work of Yibrah and Tsega (2017). including identification number, sampling date, age, breed, sex, farm type, faecal consistency and farm housing (separate housing, floor type, cleaning and disinfection), provision of supplement feedstuffs to calves, colostrum feeding and history of diarrhoea, were documented in recording format. Bacteriological culturing and examination: Suspicious colonies were further subcultured in nutrient media (HiMedia, India) and aerobically incubated for 24-48 h at 37 • C. Pure colonies were sub-cultured on MacConkey agar for 24-48 h at 37 • C following a morphological evaluation with Gram staining features. In the isolation and identification of E. coli, growth on MacConkey agar was used as the primary criterion. In addition, MacConkey agar colonial features were employed to divide putatively isolated bacteria into two groups: lactose fermenter and non-lactose fermenter. E. coli colonies suspected of further sub-cultured on agar media with Eosin methyl blue (EMB) for selective identification of E. coli. The colonies that appeared greenblack with a metallic sheen, which differentiates E. coli on EMB, were chosen and kept on nutrient agar for additional biochemical analyses after 24 h. objective, all of the isolates were stained with Gram stain to detect cell shape, Gram response, and purity (100× magnification). Primary biochemical tests were also conducted via, catalase test, triple sugar iron (TSI) test, potassium hydroxide (KOH), sulphur indole motility (SIM) test and oxidation-fermentation (O-F) test. Standard bacteriological procedures were used to identify suspected E. coli colonies and purified E. coli cultures were kept in nutrient broth for subsequent identification using biochemical testing as described in Quinn et al. (2002) . Voges-Proskauer (VP) and citrate utilisation biochemical assays were used to identify E. coli isolates preliminarily after overnight incubation at 37 • C on each of the four tests (Quinn et al., 2002) . On the other hand, E. coli isolates were further cultured on Sorbitol MacConkey agar for 24-48 h at 37 • C to identify pathogenic and non-pathogenic E. coli strains. Lactose is replaced by sorbitol in sorbitol MacConkey agar. E. coli bacteria that are not pathogenic ferment sorbitol to generate acid. Because pathogenic E. coli cells are unable to ferment sorbitol, this strain grows on peptone. This raises the medium's pH, allowing the pathogenic strain to be distinguished from other E. coli strains using the medium's pH indicator (Novicki et al., 2000) . Screening test by E. coli O157 Latex agglutination test: For the screening of E. coli O157:H7, a latex agglutination test was used with a latex kit. Sorbitol-negative (clear) colonies with colony morphology similar to E. coli O157:H7 were selected and spread plated on Cefixime tellurite sorbitol MacConkey plates (CT-SMAC). After a 24-h incubation period, a single colony of the non-sorbitol fermenter was selected from sorbitol MacConkey agar and treated with latex agglutination using an E. coli O157 latex kit. For all latex agglutination tests, an isolate suspected of being E. coli O157:H7 was cultivated on nutrient agar (NA) for antibiotic susceptibility testing. Antibiotic susceptibility profiles were conducted for E. coli O157:H7 isolates using the disc diffusion method (Kirby-Bauer method) on Mueller-Hinton agar (Oxoid, England) according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2012 (CLSI, , 2014 . Pure colonies on nutrient agar were taken with a wire loop and transferred to a tube containing 5 ml of saline water and emulsified. The broth culture was incubated at 37 • C for 4 h until it achieved the 0.5 McFarland turbidity standards. A sterile cotton swab was dipped into the suspension and the bacteria were swabbed uniformly over the surface of a Muller-Hinton agar plate within a sterile safety cabinet. The plates were held at room temperature for 15 min to allow drying. Antibiotic discs with a known concentration of antibiotics were placed and the plates were incubated for 18-24 h at 37 • C. Each isolate was tested for a series of 10 antibiotic discs (Oxoid, Eng- During this study, the samples were collected from diarrhoeic calves in an aseptic manner. Almost all calves showed disease signs such as elevated temperature, depression, dehydration, reduced suckling reflex, rough hair coat, loss of weight, weakness, soiling of the hindquarter and tail with diarrhoeic faeces. Out of 68 faecal samples collected, 47 (69.1%) of the isolates were E. coli positive. Out of 47 positive isolates, 46.8% (22/47) were E. coli O157:H7 isolates since the isolates were not able to ferment sorbitol that showed colourless colonies, and the isolates were also tested for latex agglutination using a latex kit for the screening of E. coli O157:H7 (Figure 2 ). In the present study, about 49%, 45% and 6% of the E. coli isolates were isolated from diarrhoeic calves located in medium-, small-and large-scale dairy farms, respectively. The isolation of E. coli was not significantly correlated with either the medium-(p = 0.804) or large farm type (p = 0.331). However, there was a higher negative correlation between the isolation of E. coli O157: H7 and medium (r = −0.03, 95% CI: −0.27 to −0.21) and large-sized farm types (r = −0.16, 95% CI: −0.50 to −0.17), while small-sized farms held constant (Table 1) . In the present study, factors such as sex, age, breed, management system, type of diarrhoea, method of colostrum feeding, time of first colostrum feeding, duration of colostrum feeding, frequency of cleaning calf house, type of supplement, floor type and separate housing were analysed for their influence on the occurrence of E. coli from diarrhoeic calves. The frequency of calf house cleaning, type of supplement provided and method of colostrum feeding were significantly correlated (p < 0.05) with the occurrence of E. coli from diarrhoeic calves. The cleaning of the calf's house every day was significantly correlated with the isolation of E. coli from diarrhoeic calves (p ≤ 0.001). However, there was a significantly higher negative correlation between the isolation of E. coli and cleaning of the calf house every day (r = −0.47, 95% CI: −0.71 to −0.23). About 68% of the dairy farm owners provided milk for their calf using hands or buckets and this had a significantly positive correlation with the occurrence of E. coli (r = 0.28, 95% CI: 0.054-0.51). Moreover, calves that were provided with a mixture of milk and other concentrate feedstuffs were infected with E. coli In the present study, 22 E. coli O157:H7 isolates were tested against 10 commonly used antibiotics using the disc diffusion method (Kirby- In this research, the isolation and identification of Escherichia coli O157: H7 from diarrhoeic calves were performed using standard bacteriological procedures and different precipitating factors that were responsible for the occurrence of the disease were assessed through a questionnaire survey. Furthermore, the isolates of E. coli O157: H7 were tested against different antibiotic discs. According to various study findings, calf diarrhoea was found to be a major health issue in dairy farms, causing high mortality and morbidity in calves. Calf diarrhoea has also resulted in significant economic losses, including money invested in care/treatment, loss of genetic potential due to the loss of calves and farmers' inability to invest in higher-priced semen in the face of calf mortality. It has also hampered proper heifer replacement, which affects dairy farmers' ability to increase productivity by encouraging them to cull low-producing cows selectively (Fubini & Divers, 2008; Hadgu et al., 2021; Radostits et al., 2007; Svensson et al., 2003) . In the current study, out of 68 diarrhoeic sampled calves, the isolation of Escherichia coli was 69.1% (47/68). This result was comparable with other previous research findings of Yeshiwas and Fentahun (2017) methods, environmental quality and inadequate sanitation, which also causes pathogenic strains to grow up in the ecosystem of young animals. Furthermore, a high dose of pathogenic E. coli could be enough to suppress colostral immunity (Quinn et al., 2002; Radostits et al., 2007) . The phenotypic detection of E. coli from most other serotypes was based on its inability to ferment sorbitol sugar on sorbitol MacConkey (SMAC) agar and a latex agglutination test using a latex kit for the screening of E. coli O157:H7. The present study also revealed that out of positive E. coli O157:H7 isolates, 46.8% (22/47) were not able to ferment sorbitol, which showed colourless colonies. The current study finding was much higher than the previous study report of Lee and Choi (2006) , who reported 4% ( In the present study, some factors were investigated for their association with the occurrence of E. coli from calves showing diarrhoea. Accordingly, factors such as the frequency of calf house cleaning, type of supplement provided, and method of colostrum feeding were significantly correlated (p < 0.05) with the occurrence of E. coli from diarrhoeic calves. However, isolation of E. coli was not directly significantly correlated (p > 0.05) with age, sex, type of diarrhoea, breed, management system, method of colostrum feeding and first colostrum feeding time. The current study revealed that the cleaning of the house of calves every day was significantly correlated with the isolation of E. coli from diarrhoeic calves (p ≤ 0.001). However, there was a significantly higher negative correlation between the isolation of E. coli and the cleaning of the house of calves every day (r = −0.47). Radostits et al. (2007) also indicated that the different stressing factors and the type of infection strain they face right after birth are responsible for numerous types of neonatal infections that are more common during their early years. In addition, young neonates below 1 week of age are the most vulnerable as their intestinal flora is not fully established compared with older age (Charles et al., 2003) . The study also revealed that the highest percentage (40.4%) of E. coli isolates were recovered from newly born calves (1-7 days age group) compared with other age categories. This study finding is in agreement with previous reports of different studies that revealed that younger calves were mostly clinically affected (Gebregiorgis & Tessema, 2016; Lorino et al., 2005; Maddox-Hyttel et al., 2006; Muktar, 2014; Muktar et al., 2015; Santın et al., 2004) . This study finding was also supported by reports of Villarroel (2009) , who noted that as the age of the calves increased, the incidence of calf diarrhoea decreased. This could be due to the days-old calves' immune system's inability to fight disease-causing agents compared to older calves (Darsema, 2008) . This finding was also consistent with the findings of Wudu (2004) , Aggernesh (2010) and Dereje (2012) , who reported that calves aged between 0 and 30 days were at great risk of calf diarrhoea, particularly during the first week. In contrast to the aforementioned research findings, Gebremedhin (2014) Debnath et al. (1995) and Mansour et al. (2014) reported that the sex of the calves has a significant effect on the calf mortality rate. In the present study, most (68.1%) dairy owners used hand or bucket systems to provide milk for their calf, and there was a significant positive correlation with the occurrence of E. coli (r = 0.28, p = 0.016). Moreover, most (74.5%) dairy owners provide a mixture of milk with other concentrate feedstuffs as a feed supplement for their calves. This had a significantly positive correlation with the occurrence of E. coli (r = 0.43, p = 0.002). This might have contributed to the decreased colostrum transfer that provides better passive immunity. In their first week of life, calves have a passive immune system as well as receptors for E. coli adhesions (Villarroel, 2009) . As Stoltenow and Vincent (2003) stated that the high risk of diarrhoea in calves could be due to inadequate passive transfer of colostral immunity. The research findings of Trotz-Williams et al. (2007) and Lorino et al. (2005) also revealed that delayed colostrum intake, especially in the first 6 h of age, predisposes the calf to a higher risk of E. coli prevalence. Furthermore, Olsson et al. (1993) found that with every hour of delay in the first 12-h colostrum feeding, the risk of calf infection increased by 10%. In contrast, in our investigation, the calves that obtained colostrum within the first 12 h (78.7%) had a higher degree of calf diarrhoea than calves who obtained colostrum between 12 and 24 h (12.8%). This variation might be due to calves feeding on colostrum through hand and bucket, poor sanitation of the equipment and barns. This finding was supported by Shiferaw et al. (2002) , who reported that the microenvironment hygiene of the farm has a great effect on the occurrence of calf mortality and morbidity in Holeta, Ethiopia and Bendali et al. (1999) , who stated that unclean calf barns might be linked with a higher risk of calf scour in southwest, France. Additionally, calves with irregular bedding changes, inadequate living conditions and insufficient sanitation have an elevated chance of morbidity (Charles et al., 2003; Perez et al., 1990) . Antibiotic use is an important factor in maintaining human and animal health worldwide (World Health Organization, 2014). Recently, the development of antibiotic resistance in most bacterial species has become a serious threat to global public health (Acar & Moulin, 2013; Heuer et al., 2006; Merle et al., 2012) . According to the current study, E. coli O157:H7 isolates were susceptible to gentamycin, ciprofloxacin, ceftriaxone, trimethoprimsulphamethoxazole, streptomycin and chloramphenicol. The sensitivity of the isolates to gentamycin was comparable with that of the Ababu et al. (2020) , who stated that all isolates were highly susceptible to the list of antibiotic discs. The isolates' chloramphenicol sensitivity matched the findings of Guerra et al. (2006) , who found that most bacteria isolated from diarrhoeal calves were chloramphenicol susceptible. In contrast, Abdullah et al. (2013) and Ahmad et al. (1986) reported that E. coli O157:H7 isolates were resistant to chloramphenicol. In this study, E. coli isolates were also sensitive to ciprofloxacin, Yenehiwot (2008) , whose isolated bacteria were highly susceptible to ciprofloxacin. The current research revealed that E. coli O157:H7 isolates were susceptible to streptomycin, which was in contrast to the report of Ababu et al. (2020) , who stated that E. coli O157:H7 isolates were highly resistant to streptomycin. In the present research, E. coli isolates were resistant to tetracycline, kanamycin and amoxicillin. The pres-ence of resistance against kanamycin is in agreement with the previous findings of Hiko et al. (2008) , Minda Asfaw and Shimelis (2021) and Joon and Kaura (1993) , whose isolated bacteria were less sensitive to kanamycin. Nonetheless, this was against the report of Tassew et al. (2010) and Taye et al. (2013) , in which all the E. coli isolates were found to be susceptible to kanamycin. The resistance of the isolates to tetracycline was comparable with the report of Ababu et al. (2020) , who stated that isolated E. coli species were resistant to tetracycline. This variation might be due to sample size variation, sample type used, laboratory procedures and the number of antibiotics (n = 10) used during the current study compared to antibiotics used (7-8) in other studies conducted in Ethiopia. The difference may be because of the expression of the resistance gene code via the pathogen, which is correlated with existing and emerging isolated features of various agroecological aspects (Reuben & Owuna, 2013) . The finding of high resistance of E. coli O157:H7 isolates to amoxicillin agreed with the previous results of Abdullah et al. (2013) , Abd-Elrahman (2011), Ansari et al. (2014) , Edrington et al. (2006) and Nazir and Hussain (2007) . The high resistance of these drugs in Gramnegative bacteria might be due to the transfer of resistance genes from Gram-positive bacteria of β-lactamase genes. Al-Assil et al. (2013) also explained that among the 25 E.coli isolates, the most prevalent βlactamase gene was βlaCTX-M, which was detected in all of the isolates, whereas the βlaTEM gene was found in eight isolates of E. coli. This may also be attributed to the unregulated and improper use of these agents in veterinary clinics and farms and throughout the world. This is supported by the lack of policy on antibiotic use and the accessibility of antibiotics in the region. Since E. coli is an integral part of normal faecal flora, it is a potential indicator of resistance trends in humans and animals (Werckenthin et al., 2002) . In the present study, the occurrence of E. coli O157:H7 from diarrhoeic calves was high in the dairy farms of the study area. Of the 69.1% E. coli-positive isolates, 46.8% were E. coli O157:H7 strains that could cause calf diarrhoea. Factors such as the method of colostrum feeding, hygiene of calves' barn and type of feed supplement provided were found to be significantly (p < 0.05) correlated with the occurrence of E. coli in calves. Observational and questionnaire surveys revealed that simply being aware of the benefits of colostrum feeding is insufficient; the cleanliness of the material used for colostrum administration as well as the hygiene of the calves' barn are critical for the ultimate success of E. coli O157:H7 control. Antibiotic susceptibility results revealed that most E. coli O157:H7 isolates were highly sensitive to gentamycin, chloramphenicol, ceftriaxone, ciprofloxacin trimethoprimsulphamethoxazole and streptomycin. However, some E. coli isolates were found to be resistant to tetracycline, kanamycin and amoxicillin. In conclusion, further study on the usefulness of the strain identification approach for E. coli O157:H7 strains should be carried out in comparison with PCR and serotyping. Special emphasis should be given to the time, method, and duration of colostrum feeding to the newborn calves (colostrum should be provided before 6 h in an aseptic manner). Antibiotics that are sensitive to E. coli isolates should be the drugs of choice. The treatment of this disease should be designed based on the antibiotic susceptibility pattern of the isolates. The authors would like to acknowledge all dairy cattle owners who kindly collaborate during sampling. All authors declare that there are no conflicts of interest in this work. As the study was not experimental, no ethical approval was needed. However, before collecting samples, verbal consent was also pursued from the cattle owners to take faecal samples from their cattle and adopt strict hygienic measures. HF was involved in conceptualisation, data analysis, preparing an The current study was conducted without the support of funding sources. The data will be provided upon the request of the corresponding author. The peer review history for this article is available at https://publons. 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